Fluorescent Labeling and Imaging of IL-22 mRNA-Loaded Lipid Nanoparticles

Lipid nanoparticle (LNP)-based drug delivery systems (DDSs) are widely recognized for their ability to enhance efficient and precise delivery of therapeutic agents, including nucleic acids like DNA and mRNA. Despite this acknowledgment, there is a notable knowledge gap regarding the systemic biodistribution and organ accumulation of these nanoparticles. The ability to track LNPs in vivo is crucial for understanding their fate within biological systems. Fluorescent labeling of LNPs facilitates real-time tracking, quantification, and visualization of their behavior within biological systems, providing valuable insights into biodistribution, cellular uptake, and the optimization of drug delivery strategies. Our prior research established reversely engineered LNPs as an exceptional mRNA delivery platform for treating ulcerative colitis. This study presents a detailed protocol for labeling interleukin-22 (IL-22) mRNA-loaded LNPs, their oral administration to mice, and visualization of DiR-labeled LNPs biodistribution in the gastrointestinal tract using IVIS spectrum. This fluorescence-based approach will assist researchers in gaining a dynamic understanding of nanoparticle fate in other models of interest. Key features • This protocol is developed to assess the delivery of IL-22 mRNA to ulcerative colitis sites using lipid nanoparticles. • This protocol uses fluorescent DiR dye for imaging of IL-22 mRNA-loaded lipid nanoparticles in the gastrointestinal tract of mice. • This protocol employs the IVIS spectrum for imaging.

This protocol is used in: Biomaterials (2022), DOI: 10.1016/j.biomaterials.2022.121707 Lipid nanoparticle (LNP)-based drug delivery systems (DDSs) are widely recognized for their ability to enhance efficient and precise delivery of therapeutic agents, including nucleic acids like DNA and mRNA.Despite this acknowledgment, there is a notable knowledge gap regarding the systemic biodistribution and organ accumulation of these nanoparticles.The ability to track LNPs in vivo is crucial for understanding their fate within biological systems.Fluorescent labeling of LNPs facilitates real-time tracking, quantification, and visualization of their behavior within biological systems, providing valuable insights into biodistribution, cellular uptake, and the optimization of drug delivery strategies.Our prior research established reversely engineered LNPs as an exceptional mRNA delivery platform for treating ulcerative colitis.This study presents a detailed protocol for labeling interleukin-22 (IL-22) mRNA-loaded LNPs, their oral administration to mice, and visualization of DiR-labeled LNPs biodistribution in the gastrointestinal tract using IVIS spectrum.This fluorescence-based approach will assist researchers in gaining a dynamic understanding of nanoparticle fate in other models of interest.

Background
In ulcerative colitis, interleukin-22 (IL-22) plays a crucial role by promoting mucosal healing and regulating the inflammatory response.Lipid nanoparticles (LNPs) offer a targeted delivery platform for IL-22 in this context, effectively harnessing the cytokine's therapeutic potential to address mucosal healing and inflammation precisely at the site of injury [1].Despite the increasing interest in utilizing LNPs for targeted delivery of therapeutic agents in ulcerative colitis, our understanding of their in vivo behavior is limited, hindering the clinical translation of LNPbased therapies.The tracking of LNPs in vivo can provide crucial insights into their biodistribution, migration abilities, and mechanism of action [2].Therefore, the development of efficient and sensitive techniques for labeling LNPs is highly desired.To date, several methods have been developed to unravel the in vivo dynamics of LNPs.Notably, the use of fluorescent dyes to label LNPs stands out as an effective approach for confirming successful therapeutic delivery.This highly sensitive and selective technique enables real-time monitoring and visualization of nanoparticle behavior and distribution in biological systems.However, a potential limitation of fluorescent labeling is the risk of dye leakage from nanoparticles in vivo, resulting in diminished brightness over time and the development of a background signal that may hinder accurate nanoparticle localization [3].In a prior study, we engineered LNPs loaded with IL-22 mRNA for treating ulcerative colitis, evaluating the biodistribution of DiR-labeled IL-22/LNP [4].In this protocol, we will describe the detailed process of labeling and imaging IL-22/LNP in the gastrointestinal (GI) tract using a fluorescent dye via the IVIS spectrum.In our previously published study [4], LNPs loaded with mRNA as described in this protocol displayed a distinct signal in the targeted organ (colon) and exhibited therapeutic efficacy.

B. Oral gavaging of DiR'-labeled IL-22/LNPs
1. Take healthy mice and divide them into two groups (Control group and Treated group).
2. Fast the mice for 4 h before gavage.
3. Fill DiR'-labeled IL-22/LNPs suspension into a 1 mL syringe equipped with an animal feeding needle and remove all air bubbles (Figure 5).

Figure 3 .
Figure 3. Transfer the lipid nanoparticles (LNP) suspension to a centrifugal filter